The overall aim of this research program is to develop novel, potent and highly selective inhibitors of cruzain, the major cysteine protease of Trypanosoma cruzi; falcipain-2 and -3, the major cysteine proteases of the Plasmodium falciparum trophozoite; rhodesain from Trypanosoma brucei rhodesiense; and the cathepsin B-like (Lmaicatb) and L-like cysteine proteases from Leishmania major. Efforts will be made to develop a single cysteine protease inhibitor that is effective against all of the targeted parasites. Specific aims for the next grant period include: (1) Optimization of the vinyl sulfonyl inhibitor P' substituent. (2) Development of a solid-phase synthesis of vinyl sulfonyl inhibitors. (3) Continued development and optimization of E-64 analogs. (4) Development of conformationally constrained cysteine protease inhibitors. (5) Development of heterocycle-based peptidometics as cysteine protease inhibitors. (6) Determination of enzyme-bound inhibitor conformations and active site structures via transferred Nuclear Overhauser Effect (TRNOE) and residual dipolar coupling studies. (7) NMR screening of new inhibitor scaffolds. All enzymatic and biological evaluation of the inhibitors will be performed at UCSF (McKerrow for cruzain, rhodesain and the leishmania enzymes; Rosenthal for falcipains-2 and -3). To the extent possible, the best inhibitors will be characterized by X-ray analysis of the inactivated enzyme. Inhibitor complexes of the targeted proteases that have not yielded to crystallographic analysis will be characterized by NMR methods.